title: Quantitative evaluation of protein conformation in pharmaceuticals using cross-linking reactions coupled with LC-MS/MS analysis.
abstract: The need for a simple and high-throughput method for identifying the tertiary structure of protein pharmaceuticals has increased. In this study, a simple method for mapping the protein fold is proposed for use as a complementary quality test. This method is based on cross-linking a protein using a [bis(sulfosuccinimidyl)suberate (BS(3))], followed by peptide mapping by LC-MS. Consensus interferon (CIFN) was used as the model protein. The tryptic map obtained via liquid chromatography tandem mass spectroscopy (LC-MS/MS) and the mass mapping obtained via matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy were used to identify cross-linked peptides. While LC-MS/MS analyses found that BS(3) formed cross-links in the loop region of the protein, which was regarded as the biologically active site, sodium dodecyl-sulfate polyacrylamide gel electrophoresis demonstrated that cross-linking occurred within a protein molecule, but not between protein molecules. The occurrence of cross-links at the active site depends greatly on the conformation of the protein, which is determined by the denaturing conditions. Quantitative evaluation of the tertiary structure of CIFN was thus possible by monitoring the amounts of cross-linked peptides generated. Assuming that background information is available at the development stage, this method may be applicable to process development as a complementary test for quality control.
authors.affiliation: Laboratory of Analytical and Bio-Analytical Chemistry, School of Pharmaceutical Sciences and Global COE Program, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan. hyamaguchi@agensys.com
authors.forename: Hideto
authors.initials: H
authors.lastname: Yamaguchi
authors.forename: Yutaka
authors.initials: Y
authors.lastname: Hirakura
authors.forename: Hiroki
authors.initials: H
authors.lastname: Shirai
authors.forename: Hisashi
authors.initials: H
authors.lastname: Mimura
authors.forename: Toshimasa
authors.initials: T
authors.lastname: Toyo'oka
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D002853:Chromatography, Liquid; D003432:Cross-Linking Reagents / Q000737:chemistry*; D018360:Crystallography, X-Ray; D004591:Electrophoresis, Polyacrylamide Gel; D007370:Interferon Type I / Q000032:analysis* / Q000737:chemistry; D016898:Interferon-alpha; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D010449:Peptide Mapping / Q000295:instrumentation / Q000379:methods*; D004364:Pharmaceutical Preparations / Q000032:analysis* / Q000737:chemistry; D011485:Protein Binding; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013388:Succinimides / Q000737:chemistry*; D053719:Tandem Mass Spectrometry
chemical_list: D003432:Cross-Linking Reagents; D007370:Interferon Type I; D016898:Interferon-alpha; D004364:Pharmaceutical Preparations; D011994:Recombinant Proteins; D013388:Succinimides; C050739:interferon alfacon-1; C035760:bis(sulfosuccinimidyl)suberate